CytoRADx: A High-Throughput, Standardized Biodosimetry Diagnostic System Based on the Cytokinesis-Block Micronucleus Assay.

In a large-scale catastrophe, such as a nuclear detonation in a major city, it will be crucial to accurately diagnose large numbers of people to direct scarce medical resources to those in greatest need. Currently no FDA-cleared tests are available to diagnose radiation exposures, which can lead to complex, life-threatening injuries. To address this gap, we have achieved substantial advancements in radiation biodosimetry through refinement and adaptation of the cytokinesis-block micronucleus (CBMN) assay as a high throughput, quantitative diagnostic test. The classical CBMN approach, which quantifies micronuclei (MN) resulting from DNA damage, suffers from considerable time and expert labor requirements, in addition to a lack of universal methodology across laboratories. We have developed the CytoRADx™ System to address these drawbacks by implementing a standardized reagent kit, optimized assay protocol, fully automated microscopy and image analysis, and integrated dose prediction. These enhancements allow the CytoRADx System to obtain high-throughput, standardized results without specialized labor or laboratory-specific calibration curves. The CytoRADx System has been optimized for use with both humans and non-human primates (NHP) to quantify radiation dose-dependent formation of micronuclei in lymphocytes, observed using whole blood samples. Cell nuclei and resulting MN are fluorescently stained and preserved on durable microscope slides using materials provided in the kit. Up to 1,000 slides per day are subsequently scanned using the commercially based RADxScan™ Imager with customized software, which automatically quantifies the cellular features and calculates the radiation dose. Using less than 1 mL of blood, irradiated ex vivo, our system has demonstrated accurate and precise measurement of exposures from 0 to 8 Gy (90% of results within 1 Gy of delivered dose). These results were obtained from 636 human samples (24 distinct donors) and 445 NHP samples (30 distinct subjects). The system demonstrated comparable results during in vivo studies, including an investigation of 43 NHPs receiving single-dose total-body irradiation. System performance is repeatable across laboratories, operators, and instruments. Results are also statistically similar across diverse populations, considering various demographics, common medications, medical conditions, and acute injuries associated with radiological disasters. Dose calculations are stable over time as well, providing reproducible results for at least 28 days postirradiation, and for blood specimens collected and stored at room temperature for at least 72 h. The CytoRADx System provides significant advancements in the field of biodosimetry that will enable accurate diagnoses across diverse populations in large-scale emergency scenarios. In addition, our technological enhancements to the well-established CBMN assay provide a pathway for future diagnostic applications, such as toxicology and oncology.

Intramesenteric dynamic contrast pediatric MR lymphangiography: initial experience and comparison with intranodal and intrahepatic MR lymphangiography.

OBJECTIVES: To report on our initial experience with intramesenteric (IM) dynamic contrast magnetic resonance lymphangiography (DCMRL) for evaluation of the lymphatics in patients with concern for mesenteric lymphatic flow disorders and to compare IM-DCMRL with intrahepatic (IH) and intranodal (IN) DCMRL. METHODS: This is a retrospective review of imaging findings in 15 consecutive patients who presented with protein losing enteropathy (PLE) and/or ascites undergoing IM-DCMRL, IH-DCMRL, and IN-DCMRL. The IM-DCMRL technique involves the injection of a gadolinium contrast agent into the mesenteric lymphatic ducts or lymph nodes followed by imaging of the abdomen and chest with dynamic time-resolved MR lymphangiography. RESULTS: IM-DCMRL was successfully performed in 14/15 (93%) of the patients. When comparing IN-DCMRL with IM-DCMRL, there was a significant difference in the visualization of dermal backflow (p = 0.014), duodenal perfusion (p = 0.003), duodenal leak (p = 0.014), and peritoneal leak (p = 0.003). IM-DCMRL demonstrated peritoneal leak in 7 patients in contrast to IH-DCMRL which demonstrated leak in 4 patients and IN-DCMRL which did not demonstrate any peritoneal leaks. Duodenal leaks were seen by IH-DCMRL in 9 patients versus 5 with IM-DCMRL and none with IN-DCMRL. In one patient with congenital PLE, the three modalities showed different disconnected flow patterns with duodenal leak only seen by IM-DCMRL. There were no short-term complications from the procedures. CONCLUSIONS: IM-DCMRL is a feasible imaging technique for evaluation of the mesenteric lymphatics. In certain mesenteric lymphatic flow abnormalities, such as PLE and ascites, this imaging may be helpful for diagnosis and the planning of interventions and warrants further studies. KEY POINTS: • Intramesenteric dynamic contrast magnetic resonance lymphangiography (IM-DCMRL) is a new imaging technique to evaluate mesenteric lymphatic flow disorders such as ascites. • IM-DCMRL is able to image lymphatic leaks in patients with ascites and protein losing enteropathy not seen with intranodal (IN-DCMRL) imaging.

Antibody-based methods for the measurement of α-synuclein concentration in human cerebrospinal fluid - method comparison and round robin study.

α-Synuclein is the major component of Lewy bodies and a candidate biomarker for neurodegenerative diseases in which Lewy bodies are common, including Parkinson's disease and dementia with Lewy bodies. A large body of literature suggests that these disorders are characterized by reduced concentrations of α-synuclein in cerebrospinal fluid (CSF), with overlapping concentrations compared to healthy controls and variability across studies. Several reasons can account for this variability, including technical ones, such as inter-assay and inter-laboratory variation (reproducibility). We compared four immunochemical methods for the quantification of α-synuclein concentration in 50 unique CSF samples. All methods were designed to capture most of the existing α-synuclein forms in CSF ('total' α-synuclein). Each of the four methods showed high analytical precision, excellent correlation between laboratories (R2 0.83-0.99), and good correlation with each other (R2 0.64-0.93), although the slopes of the regression lines were different between the four immunoassays. The use of common reference CSF samples decreased the differences in α-synuclein concentration between detection methods and technologies. Pilot data on an immunoprecipitation mass spectrometry (IP-MS) method is also presented. Our results suggest that the four immunochemical methods and the IP-MS method measure similar forms of α-synuclein and that a common reference material would allow harmonization of results between immunoassays.

Migration Phenotype of Brain-Cancer Cells Predicts Patient Outcomes.

Glioblastoma multiforme is a heterogeneous and infiltrative cancer with dismal prognosis. Studying the migratory behavior of tumor-derived cell populations can be informative, but it places a high premium on the precision of in vitro methods and the relevance of in vivo conditions. In particular, the analysis of 2D cell migration may not reflect invasion into 3D extracellular matrices in vivo. Here, we describe a method that allows time-resolved studies of primary cell migration with single-cell resolution on a fibrillar surface that closely mimics in vivo 3D migration. We used this platform to screen 14 patient-derived glioblastoma samples. We observed that the migratory phenotype of a subset of cells in response to platelet-derived growth factor was highly predictive of tumor location and recurrence in the clinic. Therefore, migratory phenotypic classifiers analyzed at the single-cell level in a patient-specific way can provide high diagnostic and prognostic value for invasive cancers.

Pre-exposure of human adipose mesenchymal stem cells to soluble factors enhances their homing to brain cancer.

Recent research advances have established mesenchymal stem cells (MSCs) as a promising vehicle for therapeutic delivery. Their intrinsic tropism for brain injury and brain tumors, their lack of immunogenicity, and their ability to breach the blood-brain barrier make these cells an attractive potential treatment of brain disorders, including brain cancer. Despite these advantages, the efficiency of MSC homing to the brain has been limited in commonly used protocols, hindering the feasibility of such therapies. In the present study, we report a reproducible, comprehensive, cell culture-based approach to enhance human adipose-derived MSC (hAMSC) engraftment to brain tumors. We used micro- and nanotechnological tools to systematically model several steps in the putative homing process. By pre-exposing hAMSCs to glioma-conditioned media and the extracellular matrix proteins fibronectin and laminin, we achieved significant enhancements of the individual homing steps in vitro. This homing was confirmed in an in vivo rodent model of brain cancer. This comprehensive, cell-conditioning approach provides a novel method to enhance stem cell homing to gliomas and, potentially, other neurological disorders.

Glucose-6-phosphatase is a key metabolic regulator of glioblastoma invasion.

UNLABELLED: Glioblastoma (GBM) remains the most aggressive primary brain cancer in adults. Similar to other cancers, GBM cells undergo metabolic reprogramming to promote proliferation and survival. Glycolytic inhibition is widely used to target such reprogramming. However, the stability of glycolytic inhibition in GBM remains unclear especially in a hypoxic tumor microenvironment. In this study, it was determined that glucose-6-phosphatase (G6PC/G6Pase) expression is elevated in GBM when compared with normal brain. Human-derived brain tumor-initiating cells (BTIC) use this enzyme to counteract glycolytic inhibition induced by 2-deoxy-d-glucose (2DG) and sustain malignant progression. Downregulation of G6PC renders the majority of these cells unable to survive glycolytic inhibition, and promotes glycogen accumulation through the activation of glycogen synthase (GYS1) and inhibition of glycogen phosphorylase (PYGL). Moreover, BTICs that survive G6PC knockdown are less aggressive (reduced migration, invasion, proliferation, and increased astrocytic differentiation). Collectively, these findings establish G6PC as a key enzyme with promalignant functional consequences that has not been previously reported in GBM and identify it as a potential therapeutic target. IMPLICATIONS: This study is the first to demonstrate a functional relationship between the critical gluconeogenic and glycogenolytic enzyme G6PC with the metabolic adaptations during GBM invasion.

Matrix nanotopography as a regulator of cell function.

The architecture of the extracellular matrix (ECM) directs cell behavior by providing spatial and mechanical cues to which cells respond. In addition to soluble chemical factors, physical interactions between the cell and ECM regulate primary cell processes, including differentiation, migration, and proliferation. Advances in microtechnology and, more recently, nanotechnology provide a powerful means to study the influence of the ECM on cell behavior. By recapitulating local architectures that cells encounter in vivo, we can elucidate and dissect the fundamental signal transduction pathways that control cell behavior in critical developmental, physiological, and pathological processes.

Restoration of growth phenotypes of Escherichia coli DH5alpha in minimal media through reversal of a point mutation in purB.

A point mutation (E115K) resulting in slower growth of Escherichia coli DH5alpha and XL1-Blue in minimal media was identified in the purB gene, coding for adenylosuccinate lyase (ASL), through complementation with an E. coli K-12 genomic library and serial subcultures. Chromosomal modification reversing the mutation to the wild type restored growth phenotypes in minimal media.